Neuronal apoptosis plays an essential role in brain development and contributes to neuronal loss under various neurodegenerative conditions. Genetic studies established molecular mechanisms responsible for initiation and progression of apoptosis. A critical role in this process was attributed to members of the caspase family. Caspase-3 appears to be a major effector in neuronal apoptosis. While being a common feature of developing CNS, apoptosis is atypical for mature mammalian brain under normal physiological conditions, and recent reports provide evidence that neuronal susceptibility to injury-induced apoptosis also markedly declines during brain maturation. Our data link the developmental decrease in apoptotic potential to transcriptional repression of the caspase-3 gene. With this background our objective was to identify essential basic mechanisms that underlie the silencing of this gene in mature brain. We have identified, cloned and sequenced the rat caspase-3 gene promoter, examined function of its regulatory elements, and identified the corresponding promoter of the human caspase-3 gene. Expression profiling of the essential transcription factors, which regulate caspase-3 transcription, did not reveal significant changes in their expression during brain maturation, suggesting that other mechanisms are involved in developmental regulation of caspase-3 expression. Promoter regions of rat and human caspase-3 genes have common dense CpG islands surrounding corresponding major transcription start sites. These regions include several consensus Sp1 binding sites essential for caspase-3 transcription. Inhibition of transcription initiation of genes with such promoter structures is generally associated with epigenetic regulation by hypermethylation of GpG islands, and a role for DNA methylation in developmental regulation of gene expression has been suggested by several recent studies. The goal of this proposal is to evaluate a role for DNA methylation of the caspase-3 promoter in regulation of neural susceptibility to caspase-dependent cell death. Our working hypothesis is that methylation of essential regulatory regions of this gene results in downregulation of its expression in neural cells, which subsequently leads to the repression of apoptotic potential during brain development. We propose two Specific Aims: (1) To evaluate an extent of DNA methylation of the caspase-3 gene promoter and associated histone modifications in rat brain and primary neuronal cultures as functions of developmental age; and (2) To examine the effect of DNA methylation on the caspase-3 gene expression and apoptotic potential in primary neuronal cultures. This study may result in identification of a novel endogenous neuroprotective mechanism that may have an important impact on improvement of existing therapeutic strategies to treat certain neurodegenerative conditions, such as perinatal ischemia and brain trauma, where the contribution of caspase-dependent apoptosis to neuronal loss is highly significant. [unreadable] [unreadable]